The 5' flanking regions of the genes (U1 and U2) encoding the human U1 and U2 small nuclear RNAs (snRNAs) each contain sequences that bind specifically to the simian virus (SV40) large tumor antigen (T.Ag). Substitution of these sites with sequences that lack T.Ag-binding sites did not block accumulation of U1 or U2 snRNA in a variety of cell types, but deletion of these regions resulted in the total loss of expression. Thus, these sequences may serve only a spacing function, and the T.Ag-binding sites appear not to be necessary for expression. However, coexpression of T.Ag markedly reduced expression of a U1 gene containing a high-affinity T.Ag-binding site (from the SV40 genome) in place of the U1 T.Ag-binding site. In contrast, coexpression of T.Ag enhanced synthesis of U2, but not U1, snRNA, independent of the presence of the T.Ag-binding sites. Thus, while the consensus T.Ag-binding sites within the U1 and U2 promoter regions do not appear to influence expression, the binding of SV40 T.Ag to a high-affinity site can lead to significant repression of a strong snRNA promoter, and T.Ag can enhance expression of another in the absence of a known binding site.